Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.600227
Title: The nature of protein interactions mediating co-stimulatory signalling in the immune system, involving PD-1
Author: Cheng, Xiaoxiao
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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Abstract:
PD-l , a costimulatory receptor expressed by T -cells, B-cells, NKT cells and monocytes, has emerged as one of the most potent inhibitory molecules in the immune system, and a very promising therapeutic target. The biophysical properties of the interactions of PD-i with its two ligands PD-Lt and PD-L2, however, are incompletely characterized. The question of why there are two ligands for PD-l is also unanswered. An unexpected interaction between PD-Lt and B7- 1, which also binds to other costimulatory receptors, has further complicated the interpretation of the functions of these molecules. In this thesis , experiments were undertaken to more fully characterize the interactions of PD-l within the wider context of costimulatory interactions involving T cells. Monomeric forms of PD-l, PD-Lt, PD-L2 and B7-1 were produced in order to re-analyze their interactions using surface plasmon resonance-based assays. The three- to four-fold greater affinity of PD-L2 versus PD-Ll for PO-l in humans (PD-IIPO-Ll- 7.8 μM, PO-IIPO-L2 -2.2μM) was principally due to the three-fold smaller dissociation rate for PO-L2 binding. The affinity of PO-Ll with B7-1 was much weaker than previously reported, and the interactions of PO-l with its ligands in the murine system had similar affinities, albeit ones considerably weaker than the respective human interactions. The thermodynamic properties of the PD-l system were characterized using two different approaches, van't Hoff analysis and isothermal calorimetry, and this showed that the PO-IIPO-Ll interaction is driven entropically, whereas for PD-I and PD-L2 binding enthalpy is dominant. The ΔCp values for both interactions were similar, with a slightly more negative value obtained for PO-IIPO-Ll than for PO-IIPD-L2, and consistent with binding involving the burial of relatively large hydrophobic surfaces. Comparison of the binding surface residues perturbed during ligand binding using nuclear magnetic resonance (NMR)-based analyses showed that the binding foot-print of PD-Ll is larger than that of PD-L2, even though PD-L2 binds PD-l more strongly. Comparison of the NMR data with the published crystal structures of murine PD- I complexed with human PD-Ll and mouse PD-L2 suggested that the human and mouse interactions differ in detail. Mathematical simulations based on the affinity and kinetic data, and on rigorous expression data revealed an unexpectedly limited contribution of PD-L2 to PD-I ligation during interactions of activated T-cells with mature dendritic cells (mDCs): PD-I engaged more than four-fold fewer PD-L2 than PD-Ll molecules due to the low expression of PD-L2 on mDCs. These observations suggest that the function of PD-L2 is not to enhance human PD-l engagement but to provide qualitatively different signals. The simulations also implied that the B7-IIPD-Ll interaction might have limited impact in the presence of CTLA-4, CD28 and PD-I. Finally, a novel method for identifying new receptor ligands suggested that there are unlikely to be high affinity ligands for several B7-, PD-Ll- and PD-L2-related proteins, raising the possibility that these proteins have ligand-independent functions. These findings provide a new, more rigorous structural and biophysical framework for interpreting the functions of PD-I.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.600227  DOI: Not available
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